Mechanism of amyloid β-protein dimerization determined using single-molecule AFM force spectroscopy.

Aβ42 and Aβ40 are the two primary alloforms of human amyloid β-protein (Aβ). The two additional C-terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single-molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C-termini, the N-terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N-terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N-terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments

Mechanism of amyloid β-protein dimerization determined using single-molecule AFM force spectroscopy.

Aβ42 and Aβ40 are the two primary alloforms of human amyloid β-protein (Aβ). The two additional C-terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single-molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C-termini, the N-terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N-terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N-terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments

C-Terminal Turn Stability Determines Assembly Differences between Aβ40 and Aβ42

Abstract Oligomerization of the amyloid β-protein (Aβ) is a seminal event in Alzheimer's disease. Aβ42, which is only two amino acids longer than Aβ40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36-Gly37 in Aβ42 that is not present in Aβ40. The dihedral angles of residues 36 and 37 in an Ile31-Ala42 peptide were consistent with β-turns, and a β-hairpin-like structure was indeed observed that was stabilized by hydrogen bonds and by hydrophobic interactions between residues 31-35 and residues 38-42. In contrast, Aβ(31-40) mainly existed as a statistical coil. To study the system experimentally, we chemically synthesized Aβ peptides containing amino acid substitutions designed to stabilize or destabilize the hairpin. The triple substitution Gly33Val-Val36Pro-Gly38Val ("VPV") facilitated Aβ42 hexamer and nonamer formation, while inhibiting formation of classical amyloid-type fibrils. These assemblies were as toxic as were assemblies from wild-type Aβ42. When substituted into Aβ40, the VPV substitution caused the peptide to oligomerize similarly to Aβ42. The modified Aβ40 was significantly more toxic than Aβ40. The double substitution D-Pro36-L-Pro37 abolished hexamer and dodecamer formation by Aβ42 and produced an oligomer size distribution similar to that of Aβ40. Our data suggest that the Val36-Gly37 turn could be the sine qua non of Aβ42. If true, this structure would be an exceptionally important therapeutic target

Dimer Formation Enhances Structural Differences between Amyloid β-Protein (1–40) and (1–42): An Explicit-Solvent Molecular Dynamics Study

Amyloid -protein (A) is central to the pathology of Alzheimer's disease. A 5% difference in the primary structure of the two predominant alloforms, A and A, results in distinct assembly pathways and toxicity properties. Discrete molecular dynamics (DMD) studies of A and A assembly resulted in alloform-specific oligomer size distributions consistent with experimental findings. Here, a large ensemble of DMD–derived A and A monomers and dimers was subjected to fully atomistic molecular dynamics (MD) simulations using the OPLS-AA force field combined with two water models, SPCE and TIP3P. The resulting all-atom conformations were slightly larger, less compact, had similar turn and lower -strand propensities than those predicted by DMD. Fully atomistic A and A monomers populated qualitatively similar free energy landscapes. In contrast, the free energy landscape of A dimers indicated a larger conformational variability in comparison to that of A dimers. A dimers were characterized by an increased flexibility in the N-terminal region D1-R5 and a larger solvent exposure of charged amino acids relative to A dimers. Of the three positively charged amino acids, R5 was the most and K16 the least involved in salt bridge formation. This result was independent of the water model, alloform, and assembly state. Overall, salt bridge propensities increased upon dimer formation. An exception was the salt bridge propensity of K28, which decreased upon formation of A dimers and was significantly lower than in A dimers. The potential relevance of the three positively charged amino acids in mediating the A oligomer toxicity is discussed in the light of available experimental data

Complete Phenotypic Recovery of an Alzheimer's Disease Model by a Quinone-Tryptophan Hybrid Aggregation Inhibitor

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated β-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Aβ oligomerization and fibrillization, as well as the cytotoxic effect of Aβ oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Aβ while immuno-staining of the 3rd instar larval brains showed a significant reduction in Aβ accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Aβ. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease

Continuous Flow Reactor for the Production of Stable Amyloid Protein Oligomers

The predominant working hypothesis of Alzheimer's disease is that the proximate pathologic agents are oligomers of the amyloid β-protein (Aβ). "Oligomer" is an ill-defined term. Many different types of oligomers have been reported, and they often exist in rapid equilibrium with monomers and higher-order assemblies. This has made formal structure-activity determinations difficult. Recently, Ono et al. [Ono, K., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 14745-14750] used rapid, zero-length, in situ chemical cross-linking to stabilize the oligomer state, allowing the isolation and study of pure populations of oligomers of a specific order (number of Aβ monomers per assembly). This approach was successful but highly laborious and time-consuming, precluding general application of the method. To overcome these difficulties, we developed a "continuous flow reactor" with the ability to produce theoretically unlimited quantities of chemically stabilized Aβ oligomers. We show, in addition to its utility for Aβ, that this method can be applied to a wide range of other amyloid-forming proteins

β-hairpin-mediated formation of structurally distinct multimers of neurotoxic prion peptides

Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into β-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109–122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109–122 peptide has a preference for α-helical conformations, but that this peptide can also form β-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109–122 peptide (A117V, associated with familial prion disease) increases the prevalence of β-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106–126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a β-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel β-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-β structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both β-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for β-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new atomic-level model for the formation of oligomers and fibrils of the prion protein and suggest that stabilization of β-hairpin structure may enhance cellular toxicity by altering the balance between oligomeric and fibrillar protein assemblies

An evaluation of the self-assembly enhancing properties of cell-derived hexameric amyloid-β

A key hallmark of Alzheimer’s disease is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-β (Aβ) peptide. The Aβ peptide is a product of sequential cleavage of the Amyloid Precursor Protein, the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by γ-secretase activity releases Aβ of several lengths and the Aβ42 isoform in particular has been identified as being neurotoxic. The misfolding of Aβ leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric Aβ42 and show its assembly enhancing properties which are dependent on the Aβ monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers